PROJECT SUMMARY Breast cancer is the first most frequently diagnosed cancer and the second most common cause of death in women worldwide. Patients with breast-to-brain metastases (BBMs) from stage IV breast carcinoma have poor prognosis, neurological degeneration, <10-month survival, and account for 10% of all breast cancer related deaths. Metastases arise in the brain following hematological spread of circulating mesenchymal-type cells from primary breast tumors. Competent tumor cell ?seeds?, which can adapt to the unique brain microenvironments or ?soil?, will then colonize and establish metastatic tumors. We recently showed that BBMs exhibit neuronal properties, become GABAergic, and can utilize the neurotransmitter gamma-Aminobutyric acid (GABA) as an onco-metabolite. Our current results show that BBMs express neuron-specific markers of synaptic connectivity/plasticity, indicating that these tumors mimic the neuronal properties for possible cross-talk with the brain microenvironment. Accumulating evidence underlines the importance of the brain microenvironment in the establishment and progression of metastatic cancers. The cerebrospinal fluid (CSF) produced by choroid plexus cells (CP) bathes the brain and spine parenchyma, analogous to ?water running through rice field? --and thus serves as an initial milieu to the brain for circulating breast cancer cells that reach the central nervous system. Although the role of the blood-brain-barrier (BBB) in breast-to-brain metastasis has been widely studied, the contribution of the choroid plexus, the blood-CSF-barrier, and the CSF as a microenvironmental niche has been largely unexplored. Our preliminary data suggest that breast cancer exposure to the choroid/CSF microenvironment results in loss of epithelial to mesenchymal transition (EMT) and induction of synaptic plasticity in BBM cells. We further show that when BBMs are seeded in the CSF, there is a global increase in the brain barrier- permeability. We hypothesize that the choroid/CSF microenvironment contributes as a gateway for colonization, and establishment of synaptic plasticity in breast-to-brain metastases. Using unique strengths of patient-derived surgically resected BBM xenografts and Reeler transgenic mouse model (normally used to study synaptic plasticity in neurodevelopment), we will investigate the potential roles of the CP/CSF tumor microenvironment: Specific Aim 1, as a non-neuronal source of GABA that can be used as a biomarker for non-invasively early detection of BBMs; Specific Aim 2, facilitating induction of brain synaptic plasticity in BBMs; and Specific Aim 3, in impacting permeability of the blood- brain-barrier and facilitating further metastasis through systemic circulation. The current proposal will shed further light on understanding how metastatic breast cancer cells adapt to their neural niche and open avenues for the development of novel therapeutic interventions for patients with brain metastases.